Interpretive Summary: Carnosol is a phenolic compound found in the herb rosemary and has anti-cancer activity in a variety of cancers. We have previously shown that carnosol can kill leukemia cells that were established from patients with high-risk acute lymphoblastic leukemia. We tested whether carnosol improve the killing of these leukemia cells in combination with the conventional chemotherapeutic agents cytarabine, methotrexate, and vincristine that are normally used to treat this disease. Co-treatment of the leukemia cells with carnosol and these drugs was found to reduce the amount of cell death (apoptosis), but did not affect early stages of apoptosis, such as mitochondrial membrane depolarization compared to the drugs alone. Caspase-3, the enzyme responsible for downstream events in apoptosis, was significantly more activated in cells co-treated with carnosol plus the chemotherapeutic drugs than in cells treated with drugs alone. Analysis of DNA strand breaks, a late stage of apoptotic cell death, showed that carnosol inhibited DNA fragmentation induced by the chemotherapeutic agents. These data suggest that although carnosol does not interfere with early events in apoptosis, this plant-derived compound blocks or delays the late events involving DNA fragmentation that complete the cell death pathway.

Technical Abstract:
Previously, we showed that carnosol from rosemary induced apoptosis in leukemic cells derived from patients with high-risk pre-B acute lymphoblastic leukemia (ALL). In the current study, carnosol was tested for its ability to sensitize leukemia-derived cells to or synergize with conventional chemotherapeutic agents used in the treatment of high-risk ALL. As determined by propidium iodide staining of nuclei, carnosol reduced the percentage of cell death in the pre-B ALL lines SEM, RS4;11, and REH when combined with cytarabine, methotrexate, or vincristine compared to the chemotherapeutic agents alone. Using the mitochondrial selective dye JC-1, we showed that co-treatment of the cells with carnosol and chemotherapeutic drugs did not reduce mitochondrial membrane depolarization compared to the drug treatment alone. Time course analysis of caspase-3 activation by flow cytometry showed co-treatment with carnosol and the conventional drugs increased the activation of caspase-3 significantly above that observed for the drugs alone. Flow cytometric gating analysis showed a higher percentage of caspase-3 positive cells remained in populations displaying a viable phenotype when co-treated with carnosol and drugs. Analysis of DNA strand breaks by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling showed that carnosol delayed DNA cleavage in the cells when combined with the chemotherapeutic agents. These data show that carnosol does not inhibit early stages of apoptosis and, in fact, increases the activation of one of the terminal caspases in the apoptotic pathway. However, carnosol partially blocks the terminal events responsible for fragmentation of DNA and formation of apoptotic bodies induced by chemotherapeutic drugs.